The present studies are the first to quantify the absolute levels of both P60-katanin and spastin in the nervous system. During development, the levels of P60-katanin are many times higher than the levels of spastin, but the levels of the two proteins are not as different in the adult attributable to a notable plunge in the levels of P60-katanin. It makes sense that the total levels of the two severing proteins are higher during development because the need for microtubule transport and reorganization is presumably much greater when the axon is growing. The fact that spastin mutations in humans result in no developmental abnormalities suggests that the high levels of P60-katanin are able to fully compensate for the diminished levels of spastin. Indeed, a transgenic mouse lacking functional spastin shows no detectable flaws in development (Tarrade et al., 2006
). However, these neurons show some abnormalities when cultured, and some zebrafish neurons display abnormalities in axonal growth when spastin levels are diminished using morpholinos (Wood et al., 2006
). These observations suggest that P60-katanin is capable of compensating for spastin under most circumstances but that the two severing proteins are not completely identical with regard to their functions and how they are regulated. We are currently conducting studies to further explore this possibility. For now, our goal was to evaluate potential mechanisms underlying axonal degeneration resulting from spastin mutations.
Detailed analyses of spastin mutations identified to date do not reveal an obvious candidate pathogenic mechanism for HSP (Fink, 2006
; Schickel et al., 2007
). Most of >150 human disease-related spastin mutations are nonsense, frame-shift, or splice-site mutations that presumably encode truncated spastin molecules lacking the AAA domain. Approximately 25% of spastin mutations are missense mutations falling within the AAA cassette. Because these various mutations would produce a dysfunctional protein unable to sever microtubules, the prevalent view has been that SPG4-based HSP is most probably a loss-of-function disease, with degeneration of axons resulting from affected neurons not reaching a hypothetical threshold of microtubule severing (Svenson et al., 2001
; Errico et al., 2002
). The main purpose of our quantitative Western blot analyses was to test the viability of this scenario. Consistent with the late-onset characteristic of HSP, we found that developing neurons contain far more P60-katanin than adult neurons, and hence developing neurons would be far better equipped to compensate for reductions in functional spastin levels associated with SPG4 mutations. In light of our tissue distribution studies, the lack of degeneration of long peripheral axons observed in HSP also makes sense, because adult peripheral neurons appear to express no spastin whatsoever and hence would be dependent on P60-katanin for microtubule severing.
At present, it is unknown whether equal amounts of P60-katanin and spastin have the same or different microtubule-severing activities in the axon. With regard to P60-katanin, its severing activity can be dampened by the presence of microtubule-associated proteins on the microtubules (Buster et al., 2002
; Qiang et al., 2006
) but enhanced by the presence of a cofactor termed P80-katanin (Hartman et al., 1998
; Yu et al., 2005
). Whether or not spastin is similarly regulated remains to be tested. Whatever the relative microtubule-severing activity of the two proteins may be in vivo
, our calculations indicate no obvious scenario that can explain why a 50% loss of spastin activity would reduce the overall microtubule-severing activity in the adult spinal cord to a greater extent than in other regions of the adult or developing nervous system. Thus, the only apparent way to reconcile our data with a loss-of-function scenario for HSP is to posit that the health of particularly long CNS axons is more sensitive to relatively moderate diminutions in microtubule-severing activity than other long axons of the body. This seems unlikely, however, because enzymes are generally expressed in excess of the levels needed (see Results). Finally, it is interesting to note that the age of onset of HSP is considerably earlier in patients who bear a function-blocking mutation on one spastin gene and a non-functional-blocking mutation on the other spastin gene (Svenson et al., 2004
). Together, these observations are difficult to reconcile with a loss-of-function scenario.
An alternative scenario is suggested by our observations on the distribution, expression pattern, and properties of the M1 isoform of spastin. M1 is a much less effective severing protein than M85. M1 is even less effective than ΔMIT, indicating that the N-terminal region of M1 more strongly inhibits the severing properties of spastin than the MIT domain enhances its severing properties. M1 is virtually absent from all axons at all stages of life, except for those within the adult spinal cord, in which it accounts for 20–25% of the total spastin. This is provocative because the spinal cord is the precise location of the corticospinal tracts that degenerate during HSP. These observations led us to hypothesize that disease-related mutations in spastin could generate a cytotoxic protein but only in the case of M1. In this scenario, for the spastin protein to be cytotoxic, it would have to contain the N-terminal region specific to M1 and also be functionally compromised by a mutation or by the lack of an AAA domain. To test this hypothesis, we compared the effects of truncated versions of M1 and M85 spastin lacking the AAA domain on cultures of embryonic cortical neurons. These studies demonstrated just how detrimental M1 mutants would be if they were robustly expressed during development. Neurons induced to express the truncated M1 are slower to develop, have shorter axons, and have generally less robust morphologies. Interestingly, the truncated M85 spastin (which would correspond to the mutant spastin expressed during development in HSP patients) did not cause any developmental problems in neuronal cell cultures.
To further investigate the mechanism of HSP, we tested the effects of truncated spastin M1 and M85 on FAT. Our premise that pathogenic spastin mutants could be deleterious to FAT is based on several lines of evidence linking FAT defects to HSP pathogenesis (Fink, 2003
). Genetic evidence shows that loss-of-function mutations in a kinesin-1 gene (kinesin-1A, KIF5A) can also give rise to HSP, namely in the form of HSP termed SPG10 (Reid et al., 2002
). Kinesin-1 is the principal molecular motor driving anterograde FAT (Morfini et al., 2005
), and, hence, it is provocative that suppression of its functions would elicit essentially the same phenotype as mutations of spastin. Supporting this notion, analysis of SPG4–HSP patients show a length-dependent “dying back” pattern of axonal degeneration, which is consistent with deficits in FAT (Deluca et al., 2004
; Morfini et al., 2007b
). In our studies on squid axoplasm, perfusion of full-length spastin (M1 plus M85) showed no effect on FAT, and the same was true for the truncated M85 without the ability to sever microtubules. However, the truncated M1 polypeptide strongly inhibited FAT, indicating that it is the 8 kDa N-terminal region of M1 that elicits these deleterious effects. Notably, this domain failed to show any effect on FAT when present in the context of the full-length M1 spastin protein. One possibility is that pathogenic spastin mutations induce a conformational change that results in abnormal exposure of the 8 kDa N terminal unique to M1. Consistent with this view, intragenic polymorphisms of spastin have been found within the 8 kDa N terminus that dramatically modify the HSP phenotype (Svenson et al., 2004
). In addition, spastin is known to interact with another HSP-related protein called atlastin via the 8 kDa N-terminal region of M1 (Evans et al., 2006
; Sanderson et al., 2006
). Interestingly, recessive mutations in atlastin also lead to HSP (Zhao et al., 2001
), suggesting that the binding of atlastin or other polypeptides to the N terminal of M1 could help prevent M1-induced pathology.
It has been suggested that, at least in the case of certain spastin mutations, the dysfunctional protein could associate with microtubules and impair their ability to interact with the motor proteins that fuel FAT (McDermott et al., 2003
). We are skeptical of this idea because the endogenous levels of spastin expressed in neurons are probably much too low to adversely affect FAT simply by coating the microtubules. Indeed, in our experiments, truncated M1 inhibited FAT when perfused at <100-fold the levels of the motor proteins themselves (i.e., kinesin-1, 500 nM) (Morfini et al., 2006
). Although the precise mechanism by which pathogenic M1 inhibits FAT is not directly addressed by our experiments, it may be instructive that other neuropathogenic polypeptides similarly inhibit FAT by activating kinases and phosphatases involved in the regulation of molecular motor proteins (Morfini et al., 2002
; Pigino et al., 2003
; Szebenyi et al., 2003
). Collectively, these experimental results provide an initial set of clues that will be helpful in identifying the molecular pathway(s) by which spastin mutations may lead to axonal degeneration through a novel gain-of-function mechanism.